EP1486571B1 - Verfahren zur rekombinanten Herstellung von Polypeptiden - Google Patents

Verfahren zur rekombinanten Herstellung von Polypeptiden Download PDF

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Publication number
EP1486571B1
EP1486571B1 EP03012295A EP03012295A EP1486571B1 EP 1486571 B1 EP1486571 B1 EP 1486571B1 EP 03012295 A EP03012295 A EP 03012295A EP 03012295 A EP03012295 A EP 03012295A EP 1486571 B1 EP1486571 B1 EP 1486571B1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
fermentation
host cell
gene
inclusion bodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP03012295A
Other languages
English (en)
French (fr)
Other versions
EP1486571A1 (de
Inventor
Rainer Grünbeck
Erhard Kopetzki
Friedrich Popp
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Priority to ES03012295T priority Critical patent/ES2253604T3/es
Priority to AT03012295T priority patent/ATE312939T1/de
Priority to DE60302776T priority patent/DE60302776T2/de
Priority to DK03012295T priority patent/DK1486571T3/da
Priority to EP03012295A priority patent/EP1486571B1/de
Priority to CA002467142A priority patent/CA2467142C/en
Priority to IL161994A priority patent/IL161994A/en
Priority to MXPA04004934A priority patent/MXPA04004934A/es
Priority to TW093115974A priority patent/TWI282370B/zh
Priority to JP2004171458A priority patent/JP3783963B2/ja
Priority to CNB2004100493772A priority patent/CN1284850C/zh
Priority to KR1020040042867A priority patent/KR100614020B1/ko
Priority to US10/866,567 priority patent/US7034119B2/en
Publication of EP1486571A1 publication Critical patent/EP1486571A1/de
Application granted granted Critical
Publication of EP1486571B1 publication Critical patent/EP1486571B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the recombinant plasmid-encoded protein biosynthesis rate can be . reduced, thereby preventing or reducing an accumulation of the protein to form insoluble protein aggregates (cf., e.g., Kopetzki, E., et al., Mol. Gen. Genet. 216 (1989) 149-155; and EP 0 300 425).
  • a method for obtaining anbibodies in soluble, correctly folded form by elevated temperature is known from WO 9402608. In other instances, it is desired, however, to prepare the recombinant protein via the route of inclusion bodies.
  • Insoluble inclusion bodies are formed during recombinant expression of polypeptides in microbial host cells.
  • Inclusion bodies are refractile aggregates of protease-resistant misfolded desired protein that occur upon over-expression of the encoding gene (Misawa, S., and Kumagai, I., Biopolymers 51 (1999) 297-307).
  • the desired polypeptide accumulates as insoluble inclusion bodies. Therefore, the yield of desired polypeptide in the insoluble portion of the fermentation batch can be enhanced.
  • the duration of incubation and the incubation temperature are uncritical per se. With increasing incubation time and at higher incubation temperatures, at first a larger amount of inclusion bodies is formed. A very long duration of incubation and very high incubation temperatures, however, result in the polypeptide being changed irreversibly, for instance, by irreversible denaturation by heat.
  • the fermentation temperature can be in the usual range for recombinant production of polypeptides in microbial host cells.
  • the fermentation temperature is preferably 30°C or lower, especially if an N-terminal processing (e.g., N-terminal cleavage of methionine of the desired polypeptide) is desired.
  • N-terminal processing e.g., N-terminal cleavage of methionine of the desired polypeptide
  • Such an N-terminal processing is performed enzymatically by endogenous enzymes of the host cell during fermentation.
  • the range of temperature is between 18 and 28°C, and most preferably, between 22 and 26°C.
  • the RBSII T-repeat gene which is about 690 bp long and flanked by a singular EcoRI and CelII restriction endonuclease cleavage site was prepared from oligonucleotides by chemical synthesis.
  • the double-stranded RBSII T-repeat gene was assembled by annealing and ligation of the oligonucleotides and subsequently cloned as an EcoRI/CelII fragment of a length of 691 bp into an E.coli plasmid.
  • the desired plasmid was designated pT-repeat.
  • the predetermined DNA sequence of the cloned RBSII T-repeat gene was confirmed by DNA sequencing.
  • the plasmid pBRori-URA3-LacI-RFN-Edel is a vector for the expression of interferon- ⁇ -2a (IFN- ⁇ -2a) in E.coli. It is based on the IFN- ⁇ -2b expression plasmid OripBR-URA3-EK-IFN (U.S. Patent No. 6,291,245).
  • pBRori-URA3-LacI-RFN-Edel differs from OripBR-URA3-EK-IFN by an additionally present lacI repressor gene and an IFN- ⁇ -2a gene instead of an IFN- ⁇ -2b gene.
  • IFN- ⁇ -2a and IFN- ⁇ -2b differ only by one amino acid at position 21 (Lys21Arg exchange).
  • the cells were analyzed in regard to the expressed polypeptides (soluble portion versus insoluble portion) as described in Example 5.
  • Polypeptide Standard fermentation at 25°C Incubation at 45°C/1 h Supernatant (soluble) Pellet (insoluble) Supernatant (soluble) Pellet (insoluble) IFN- ⁇ -2a > 95% ⁇ 5% ⁇ 2% > 98% T-repeat 40% 60% ⁇ 2% > 98% Analogous results were obtained under the post-incubation conditions 42°C / 1.5 h, 50°C / 1h. or 50°C / 45 min.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (4)

  1. Verfahren zur rekombinanten Herstellung eines gewünschten Polypeptids durch Exprimieren einer für das Polypeptid codierenden Nukleinsäure in einer mikrobiellen Wirtszelle, Bilden von Einschlusskörpern die das Polypeptid enthalten in dem Cytoplasma der Wirtszelle und Isolieren, Solubilisieren und Naturieren das Polypeptids,
    dadurch gekennzeichnet, dass nach Fermentation die Wirtszelle oder der Inhalt der Wirtszelle bei einer Temperatur von 40 °C oder höher für wenigstens 10 Minuten inkubiert wird und anschließend unlösliches Polypeptid aus der Wirtszelle isoliert wird.
  2. Verfahren nach Anspruch 1,
    dadurch gekennzeichnet, dass die Fermentation bei einer Temperatur von 30 °C oder niedriger durchgeführt wird.
  3. Verfahren nach Anspruch 1 oder 2,
    dadurch gekennzeichnet, dass die Inkubation für 10 bis 180 Minuten durchgeführt wird.
  4. Verfahren nach einem der Ansprüche 1 bis 3,
    dadurch gekennzeichnet, dass die Inkubation bei einer Temperatur von 40 °C bis 60 °C durchgeführt wird.
EP03012295A 2003-06-12 2003-06-12 Verfahren zur rekombinanten Herstellung von Polypeptiden Expired - Lifetime EP1486571B1 (de)

Priority Applications (13)

Application Number Priority Date Filing Date Title
ES03012295T ES2253604T3 (es) 2003-06-12 2003-06-12 Metodo para la produccion recombinante de polipeptidos.
AT03012295T ATE312939T1 (de) 2003-06-12 2003-06-12 Verfahren zur rekombinanten herstellung von polypeptiden
DE60302776T DE60302776T2 (de) 2003-06-12 2003-06-12 Verfahren zur rekombinanten Herstellung von Polypeptiden
DK03012295T DK1486571T3 (da) 2003-06-12 2003-06-12 Fremgangsmåde til rekombinant fremstilling af polypeptider
EP03012295A EP1486571B1 (de) 2003-06-12 2003-06-12 Verfahren zur rekombinanten Herstellung von Polypeptiden
CA002467142A CA2467142C (en) 2003-06-12 2004-05-12 Improved method for the recombinant production of polypeptides
IL161994A IL161994A (en) 2003-06-12 2004-05-13 Method for the production of insoluble polypeptide expressed in microbial host
MXPA04004934A MXPA04004934A (es) 2003-06-12 2004-05-24 Metodo mejorado para la produccion recombinante de proteinas.
TW093115974A TWI282370B (en) 2003-06-12 2004-06-03 Improved method for the recombinant production of polypeptides
JP2004171458A JP3783963B2 (ja) 2003-06-12 2004-06-09 ポリペプチドの改善された組換え的産生方法
CNB2004100493772A CN1284850C (zh) 2003-06-12 2004-06-10 重组生产多肽的改进方法
KR1020040042867A KR100614020B1 (ko) 2003-06-12 2004-06-11 폴리펩티드의 재조합 생산을 위한 향상된 방법
US10/866,567 US7034119B2 (en) 2003-06-12 2004-06-12 Method for recombinant production of polypeptides

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP03012295A EP1486571B1 (de) 2003-06-12 2003-06-12 Verfahren zur rekombinanten Herstellung von Polypeptiden

Publications (2)

Publication Number Publication Date
EP1486571A1 EP1486571A1 (de) 2004-12-15
EP1486571B1 true EP1486571B1 (de) 2005-12-14

Family

ID=33185854

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03012295A Expired - Lifetime EP1486571B1 (de) 2003-06-12 2003-06-12 Verfahren zur rekombinanten Herstellung von Polypeptiden

Country Status (13)

Country Link
US (1) US7034119B2 (de)
EP (1) EP1486571B1 (de)
JP (1) JP3783963B2 (de)
KR (1) KR100614020B1 (de)
CN (1) CN1284850C (de)
AT (1) ATE312939T1 (de)
CA (1) CA2467142C (de)
DE (1) DE60302776T2 (de)
DK (1) DK1486571T3 (de)
ES (1) ES2253604T3 (de)
IL (1) IL161994A (de)
MX (1) MXPA04004934A (de)
TW (1) TWI282370B (de)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012028525A2 (en) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Method for producing a lipid particle, the lipid particle itself and its use
WO2012028526A2 (en) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Tetranectin-apolipoprotein a-i, lipid particles containing it and its use
WO2012028523A2 (en) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Prokaryotic expression construct
WO2013026860A1 (en) 2011-08-25 2013-02-28 F. Hoffmann-La Roche Ag Shortened tetranectin-apolipoprotein a-i fusion protein, a lipid particle containing it, and uses thereof
WO2013127700A1 (en) 2012-02-29 2013-09-06 F. Hoffmann-La Roche Ag Method for reduction of 1->2 reading frame shifts
WO2014128135A1 (en) 2013-02-22 2014-08-28 F. Hoffmann-La Roche Ag Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008054673A (ja) * 2006-08-03 2008-03-13 Hokkaido Univ 組み換え蛋白質の製造方法
DE102011118029A1 (de) * 2011-06-20 2012-12-20 Universität Leipzig Modifizierte antibiotische Peptide mit variabler systemischer Freisetzung
JP6576650B2 (ja) * 2015-03-03 2019-09-18 旭化成株式会社 グルコースの溶出抑制方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9215540D0 (en) * 1992-07-22 1992-09-02 Celltech Ltd Protein expression system
US6291245B1 (en) * 1998-07-15 2001-09-18 Roche Diagnostics Gmbh Host-vector system

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012028525A2 (en) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Method for producing a lipid particle, the lipid particle itself and its use
WO2012028526A2 (en) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Tetranectin-apolipoprotein a-i, lipid particles containing it and its use
WO2012028523A2 (en) 2010-08-30 2012-03-08 F. Hoffmann-La Roche Ag Prokaryotic expression construct
WO2013026860A1 (en) 2011-08-25 2013-02-28 F. Hoffmann-La Roche Ag Shortened tetranectin-apolipoprotein a-i fusion protein, a lipid particle containing it, and uses thereof
US8791063B2 (en) 2011-08-25 2014-07-29 Hoffmann-La Roche, Inc. Shortened tetranectin-apolipoprotein A-I fusion protein, a lipid particle containing it, and uses thereof
US9139640B2 (en) 2011-08-25 2015-09-22 Hoffmann-La Roche Inc. Shortened tetranectin-apolipoprotein A-1 fusion protein, a lipid particle containing it, and uses thereof
WO2013127700A1 (en) 2012-02-29 2013-09-06 F. Hoffmann-La Roche Ag Method for reduction of 1->2 reading frame shifts
US9487576B2 (en) 2012-02-29 2016-11-08 Hoffmann-La Roche Inc. Method for reduction of 1->2 reading frame shifts
WO2014128135A1 (en) 2013-02-22 2014-08-28 F. Hoffmann-La Roche Ag Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide

Also Published As

Publication number Publication date
US20050003485A1 (en) 2005-01-06
US7034119B2 (en) 2006-04-25
TWI282370B (en) 2007-06-11
TW200510544A (en) 2005-03-16
KR20040107397A (ko) 2004-12-20
CN1572872A (zh) 2005-02-02
JP3783963B2 (ja) 2006-06-07
ATE312939T1 (de) 2005-12-15
MXPA04004934A (es) 2005-08-10
IL161994A (en) 2010-02-17
KR100614020B1 (ko) 2006-08-22
CN1284850C (zh) 2006-11-15
DE60302776D1 (de) 2006-01-19
DK1486571T3 (da) 2006-04-18
JP2005000170A (ja) 2005-01-06
IL161994A0 (en) 2005-11-20
ES2253604T3 (es) 2006-06-01
EP1486571A1 (de) 2004-12-15
CA2467142A1 (en) 2004-12-12
CA2467142C (en) 2008-01-15
DE60302776T2 (de) 2006-08-17

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